Critical-size bone defect repair using amniotic fluid stem cell/collagen constructs: Effect of oral ferutinin treatment in rats

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This study aims to evaluate the bone regeneration in a rat calvarias critical size bone defect treated with a construct consisting of collagen type I and human amniotic fluid stem cells (AFSCs) after oral administration of phytoestrogen ferutinin.

Main methods

In 12 week old male rats (n = 10), we performed two symmetric full-thickness cranial defects on each parietal region, and a scaffold was implanted into each cranial defect. The rats were divided into four groups: 1) collagen scaffold, 2) collagen scaffold + ferutinin at a dose of 2 mg/kg/5 mL, 3) collagen scaffold + AFSCs, and 4) collagen scaffold + AFSCs + ferutinin. The rats were sacrificed after 4 weeks, and the calvariae were removed, fixed, embedded in paraffin and cut into 7 μm thick sections. Histomorphometric measures, immunohistochemical and immunofluorescence analyses were performed on the paraffin sections.

Key findings

The histomorphometric analysis on H&E stained sections showed a significant increase in the regenerated area of the 4th group compared with the other groups. Immunohistochemistry performed with a human anti-mitochondrial antibody showed the presence of AFSCs 4 weeks after the transplant. Immunofluorescence analysis revealed the presence of osteocalcin and estrogen receptors (ERα and GPR30) in all groups, with a greater expression of all markers in samples where the scaffold was treated with AFSCs and the rats were orally administered ferutinin.


Our results demonstrated that the oral administration of ferutinin is able to improve the bone regeneration of critical-size bone defects in vivo that is obtained with collagen–AFSCs constructs.

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